Evaluation of four laboratory-based high-throughput SARS-CoV-2 automated antigen tests compared to RT-PCR on nasal and oropharyngeal samples.

BACKGROUND: The demand for RT-PCR testing has been unprecedented during the SARS-CoV-2 pandemic. Fully automated antigen tests (AAT) are less cumbersome than RT-PCR, but data on performance compared to RT-PCR are scarce. METHODS: The study consists of two parts. A retrospective analytical part, comparing the performance of four different AAT on 100 negative and 204 RT-PCR positive deep oropharyngeal samples divided into four groups based on RT-PCR cycle of quantification levels. In the prospective clinical part, 206 individuals positive for and 199 individuals negative for SARS-CoV-2 were sampled from either the anterior nasal cavity (mid-turbinate) or by deep oropharyngeal swabs or both. The performance of AATs was compared to RT-PCR. RESULTS: The overall analytical sensitivity of the AATs differed significantly from 42% (95% CI 35-49) to 60% (95% CI 53-67) with 100% analytical specificity. Clinical sensitivity of the AATs differed significantly from 26% (95% CI 20-32) to 88% (95% CI 84-93) with significant higher sensitivity for mid-turbinate nasal swabs compared to deep oropharyngeal swabs. Clinical specificity varied from 97% to 100%. CONCLUSION: All AATs were highly specific for detection of SARS-CoV-2. Three of the four AATs were significantly more sensitive than the fourth AAT both in terms of analytical and clinical sensitivity. Anatomical test location significantly influenced the clinical sensitivity of AATs.

[Evaluation of COVID-19 Antigen Test Kit Assay Analytical and Clinical Performance].

Objective To rapidly screen patients with novel coronavirus pneumonia (COVID-19) infection including asymptomatic ones. Method Established a rapid detection test kit, and evaluated analytical and clinical performance of it. Result The minimum limit of detection of the reagent was 9.75×102 TCID50/mL; there was no cross-reaction and interference in the high-concentration samples of 29 common respiratory pathogens tested. The diagnostic sensitivity of clinical samples was 98.56%, specificity was 99.00%, and the total coincidence rate was 98.85%; the consistency test Kappa value is 0.974 5. The stratified analysis of positive samples with different Ct values showed that the coincidence rate within each stratum was greater than 95%. Conclusion This COVID-19 antigen test kit with excellent detection performance, fast detection speed, and portable operation. It can be used as a supplementary method for existing nucleic acid detection methods for early screening of new coronavirus.

Performance Evaluation of Four Qualitative RT-PCR Assays for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019, and its rapid spread around the globe led the World Health Organization to declare it a pandemic. Laboratory diagnostics provide important information to help control virus transmission, and molecular nucleic acid amplification tests have been recognized as the gold standard for the direct detection of viral genetic material. The main aim of this study was to independently evaluate the analytical performance of four molecular assays that were designed for the detection of SARS-CoV-2 on open testing platforms under emergency use approval, namely, the COVIWOK COVID-19 RT-PCR Meril COVID-19 One-step RT-PCR Kit, the AmoyDx Novel Coronavirus (2019-nCoV) Detection Kit, the Meril COVID-19 One-step RT-PCR Kit and the NeoPlex COVID-19 Detection Kit, as alternatives to the current standard of care (SOC) assays in-country. All of the evaluated assays showed an acceptable performance, with a specificity of 100% and a sensitivity of 93.8% to 98.4%, compared to a SOC assay, with a Cohen's kappa coefficient of ≥0.9 (95% CI). In addition, the assays detected the AccuPlex reference material at 100 copies/mL, suggesting a good limit of detection. These assays provide suitable alternatives to the SOC assays that are currently available in-country, and these alternatives are acceptable for diagnostic use in South Africa. IMPORTANCE Laboratory diagnosis plays an important role in curbing the transmission of infection and reducing harmful delays in clinical and public health responses. Alternatives to the current standard of care assays for SARS-CoV-2 are important in order to overcome the challenges that are associated with global demands and supply shortages. Four molecular assays for the detection of SARS-CoV-2 that were designed for open testing platforms were evaluated in this study under emergency use approval. These assays had acceptable performance and provide suitable alternatives to the current standard of care assays that are available in-country. Their compatibilities with existing in-country amplification platforms make these assays convenient to use for diagnostic testing, both locally and globally These assays were recommended to the South African Health Products Regulatory Authority (SAHPRA) for patient care in South Africa.

Analytical performance of rapid nucleic acid detection assays and routine RT-qPCR assays for detection of SARS-CoV-2 in Shanghai, China in 2022.

Diagnostic accuracy of COVID-19 varies among different assays. In this study, the analytical performance of 1 rapid nucleic acid detection assay (Coyote assay) and 2 routine RT-qPCR assays (BioGerm assay and DaAn assay) was evaluated, using 1196 clinical samples. Disagreement in the results of 2 paired targets occurred in all 3 assays. The Coyote assay failed to detect 15 samples, and the DaAn assay failed to detect 5 samples. The Cohen's kappa coefficient was 0.970 between the BioGerm and DaAn assays, 0.907 between the Coyote and BioGerm assays, and 0.936 between the Coyote and DaAn assays. The positive percent agreement, and negative percent agreement of the Coyote assay were 84.04%, and 100%, respectively. Our study revealed that the results of the Coyote, BioGerm, and DaAn assays were highly consistent, which provided reference for the application of these assays for diagnosis of COVID-19.

Reference Interval for the Axis-Shield Clinical Chemistry Heparin-Binding Protein Assay.

The newly developed Axis-Shield clinical chemistry heparin-binding protein (HBP) assay (Axis-Shield Diagnostics Ltd., Dundee, Scotland) can be applied to fully automated platforms. We aimed to establish a reference interval (RI) of HBP using the Axis-Shield HBP assay, and to evaluate the analytical performance of this assay. An RI was established in 212 sodium citrated plasma samples using the non-parametric method (2.5th and 97.5th percentiles). Precision, linearity, and carry-over were evaluated according to the Clinical and Laboratory Standards Institute guidelines. The RI of HBP was between 5.3 ng/mL and 171.0 ng/mL, which could be applied regardless of gender and age. Percentage coefficients of variations (%CVs) of repeatability and within-laboratory precision were 4.9% and 6.3%, respectively, for low-concentration control and 1.6% and 3.0%, respectively, for high-concentration control. The linearity was excellent (coefficient of determination (R2) = 0.99), and the carry-over rate was negligible (0.05%). This is the first study to establish an RI of HBP using the newly developed and fully automated Axis-Shield HBP assay. The Axis-Shield HBP assay showed an acceptable level of analytical performance and could be used to measure HBP concentrations effectively in routine clinical practice. Further studies are awaited to evaluate the clinical utility of HBP using this automated assay.

Setting Bias Specifications Based on Qualitative Assays With a Quantitative Cutoff Using COVID-19 as a Disease Model.

OBJECTIVES: Automated qualitative serology assays often measure quantitative signals that are compared against a manufacturer-defined cutoff for qualitative (positive/negative) interpretation. The current general practice of assessing serology assay performance by overall concordance in a qualitative manner may not detect the presence of analytical shift/drift that could affect disease classifications. METHODS: We describe an approach to defining bias specifications for qualitative serology assays that considers minimum positive predictive values (PPVs) and negative predictive values (NPVs). Desirable minimum PPVs and NPVs for a given disease prevalence are projected as equi-PPV and equi-NPV lines into the receiver operator characteristic curve space of coronavirus disease 2019 serology assays, and the boundaries define the allowable area of performance (AAP). RESULTS: More stringent predictive values produce smaller AAPs. When higher NPVs are required, there is lower tolerance for negative biases. Conversely, when higher PPVs are required, there is less tolerance for positive biases. As prevalence increases, so too does the allowable positive bias, although the allowable negative bias decreases. The bias specification may be asymmetric for positive and negative direction and should be method specific. CONCLUSIONS: The described approach allows setting bias specifications in a way that considers clinical requirements for qualitative assays that measure signal intensity (eg, serology and polymerase chain reaction).

Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible.

Strategies for the development of photonic sensors for COVID-19

Beyond the optical and analytical performance of the sensor itself, the development of an optical detection tool in response to a pressing research or diagnostic need requires consideration of a host of additional factors. This talk will provide an overview of two photonic sensor systems developed for profiling the human immune response to COVID-19 infection and/or vaccination. One, focused on the design goal of high multiplexing (many targets per sensor), was built on the Arrayed Imaging Reflectometry (AIR) platform. AIR is a free-space optics technique that relies on the creation and target molecule binding-induced disruption of an antireflective coating on the surface of a silicon chip. The second method, focused on low cost and high speed, uses a small (1 x 4 mm) ring resonator photonic chip embedded in a plastic card able to provide passive transport of human samples. This “disposable photonics” platform is able to detect and quantify anti-COVID antibodies in a human sample in a minute, making it attractive for high-throughput testing applications. © 2022 SPIE

Evaluation of the analytical performance of anti-SARS-CoV-2 antibody test kits distributed or developed in Japan.

Background: With the spread of COVID-19, anti-SARS-CoV-2 antibody tests have been utilized. Herein we evaluated the analytical performance of anti-SARS-CoV-2 antibody test kits using a new reference standard prepared from COVID-19 patient sera. Methods: Fifty-seven kits in total (16 immunochromatography types, 11 ELISA types and 30 types for automated analyzers) were examined. By measuring serially diluted reference standards, the maximum dilution factor showing a positive result and its precision were investigated. Results: The measured cut-off titers varied largely depending on the antibody kit; however, the variability was small, with the titers obtained by each kit being within twofold in most cases. Conclusion: The current results suggest that a suitable kit should be selected depending on the intended purpose.

Comparison of IL-6 measurement methods with a special emphasis on COVID-19 patients according to equipment and sample type.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine associated with various diseases, including coronavirus disease (COVID-19). Although IL-6 levels can be assessed using serum samples, use of the AFIAS (Boditech Med Inc.) automated immunoassay analyzer enables quick and simple measurement of IL-6 levels in both serum and whole blood specimens. This study aimed to assess the correlation between IL-6 measurements obtained from the AFIAS IL-6 assay and Elecsys IL-6 assay (Roche Diagnostics). Additionally, utilization of the AFIAS IL-6 assay was evaluated. METHODS: The IL-6 levels from 113 serum samples quantified using two assay systems were evaluated for their degree of correlation. Meanwhile, the linearity, analytical sensitivity, and precision/reproducibility of the AFIAS IL-6 assay were also assessed. RESULTS: Quantification of IL-6 with the AFIAS IL-6 and Elecsys IL-6 assays showed excellent agreement (kappa 0.802) and were found to be correlated (y = -0.2781 + 1.068x; 95% confidence interval: 1.007-1.124). AFIAS IL-6 showed good analytical performances. IL-6 levels were significantly higher in deceased patients compared to those with non-complicated disease and those who were intubated (p = 0.002 and p < 0.0001, respectively). Finally, IL-6 levels more accurately predicted poor prognosis in patients, than did C-reactive protein (area under the curve, 0.716 vs. 0.634). CONCLUSION: The overall analytical performance of the AFIAS assay was comparable to that of the Elecsys IL-6 assay. In light of the ongoing COVID-19 pandemic, the AFIAS may be an attractive tool for measuring IL-6 levels.

Performance of the Abbott SARS-CoV-2 IgG II Quantitative Antibody Assay Including the New Variants of Concern, VOC 202012/V1 (United Kingdom) and VOC 202012/V2 (South Africa), and First Steps towards Global Harmonization of COVID-19 Antibody Methods.

In the initial stages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, a plethora of new serology tests were developed and introduced to the global market. Many were not evaluated rigorously, and there is a significant lack of concordance in results across methods. To enable meaningful clinical decisions to be made, robustly evaluated, quantitative serology methods are needed. These should be harmonized to a primary reference material, allowing for the comparison of trial data and improved clinical decision making. A comprehensive evaluation of the new Abbott IgG II anti-SARS-CoV-2 IgG method was undertaken using CLSI-based protocols. Two different candidate primary reference materials and verification panels were assessed with a goal to move toward harmonization. The Abbott IgG II method performed well across a wide range of parameters with excellent imprecision (<3.5%) and was linear throughout the positive range (tested to 38,365 AU/ml). The sensitivity (based on ≥14-day post-positive reverse transcription-PCR [RT-PCR] samples) and specificity were 98.3% (90.6% to 100.0%) and 99.5% (97.1% to 100%), respectively. The candidate reference materials showed poor correlation across methods, with mixed responses noted in methods that use the spike protein versus the nucleocapsid proteins as their binding antigen. The Abbott IgG II anti-SARS-CoV-2 measurement appears to be the first linear method potentially capable of monitoring the immune response to natural infection, including from new emerging variants. The candidate reference materials assessed did not generate uniform results across several methods, and further steps are needed to enable the harmonization process.

Evaluation of 32 rapid tests for detection of antibodies against SARS-CoV-2.

AIMS: To evaluate the analytical performance of 32 rapid tests for detection of antibodies against coronavirus SARS-CoV-2. MATERIALS AND METHODS: We used at total of 262 serum samples (197 pre-pandemic and 65 convalescent COVID-19), and three criteria to evaluate the rapid tests under standardized and optimal conditions: (i) Immunoglobulin G (IgG) specificity "good" if lower limit of the 95% confidence interval was ≥ 97.0%, "acceptable" if point estimate was ≥ 97.0%, otherwise "not acceptable". (ii) IgG sensitivity "good" if point estimate was ≥ 90.0%, "acceptable" if ≥ 85.0%, otherwise "not acceptable". (iii) User-friendliness "not acceptable" if complicated to perform or difficult to read result, otherwise "good". We also included partial evaluations of three automated immunoassay systems. RESULTS: Sensitivity and specificity varied considerably; IgG specificity between 90.9% (85.9-94.2) and 100% (97.7-100.0), and IgG sensitivity between 53.8% (41.9-65.4) and 98.5% (91.0-100.0). Combining our evaluation criteria, none of the 28 rapid tests that detected IgG had an overall performance considered "good", seven tests were considered "acceptable", while 21 tests were considered "not acceptable". Four tests detected only total antibodies and were not given an overall evaluation. IgG sensitivity and/or specificity of the automated immunoassays did not exceed that of many rapid tests. CONCLUSION: When prevalence is low, the most important analytical property is a test's IgG specificity, which must be high to minimize false positive results. Out of 32 rapid tests, none had a performance classified as "good", but seven were classified as "acceptable".

Analytical performance evaluation of five RT-PCR kits for severe acute respiratory syndrome coronavirus 2.

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.

Testing COVID-19 tests faces methodological challenges.

In battling the COVID-19 pandemic, testing is essential. The detection of viral RNA allows the identification of infected persons, whereas the detection of antibodies may reveal a response to a previous infection. Tests for coronavirus should be rigorously evaluated in terms of their analytical and clinical performance. This poses not only logistic challenges, but also methodological ones. Some of these are generic for the diagnostic accuracy paradigm, whereas others are more specific for tests for viruses. Problematic for evaluations of the clinical performance of tests for viral RNA is the absence of an independent reference standard. Many studies lack rigor in terms of the recruitment of study participants. Study reports are often insufficiently informative, which makes it difficult to assess the applicability of study findings. Attempts to summarize the performance of these tests in terms of a single estimate of the clinical sensitivity fail to do justice to the identifiable sources of the large heterogeneity in mechanisms for generating false negative results.